Trafficking of proteins and RNAs between the nucleus and the cytoplasm is a fundamental process of eukaryotic cells, which is required to segregate and coordinate the functions of the nuclear and cytoplasmic compartments. Nuclear transport is mediated by nucleocytoplasmic shuttling receptors of the karyopherin (importin/exportin) superfamily. Karyopherins interact with nucleoporins to translocate protein and RNA cargoes across the nuclear envelope, and are regulated by the small GTPase Ran. Up to now, only one class of small molecule inhibitors of the nuclear transport machinery has been described, which targets the exportin Crm1. The goal of this project is to develop and validate a high-throughput screening (HTS) assay involving permeabilized cells, to discover additional small molecule inhibitors that target the nuclear import machinery. The focus will involve the prototypical nuclear transport receptor importin beta, and two of its cargoes whose import involves different conformations of this receptor. The screen will use automated data acquisition and processing to measure the accumulation of fluorescently labeled cargoes in the nucleus of permeabilized cells, which are cultured in 96-well plates. It is expected that transfer of this assay to a 384-well format, with robotic handling of all reagents, will be straightforward. Since nuclear import requires multiple protein-protein interactions involving importin beta and Ran, the permeabilized cell screen is high content. Nonetheless, straightforward protocols for validation of hits and target identification are feasible and will be described. In a pilot study, the assay will be used to screen approximately 5000 compounds from a combinatorial library of peptidomimetic small molecules, to evaluate whether peptidomimetic libraries can yield useful inhibitors of nuclear transport. Transport of protein and RNAs between the nucleus and the cytoplasmic is critical to the proper functioning of all human cells, and can be perturbed in pathological conditions. This work is expected to provide new pharmacological tools to investigate the functioning of the nuclear- cytoplasmic transport machinery, and to contribute information that will promote the development of new avenues for treatment of human diseases. [unreadable] [unreadable] [unreadable]